DESCRIPTION (Adapted from Applicant's Description): Secretion of hormones and neurotransmitters is a multistep process by which endocrine cells and neurons communicate with their target cells. Two types of secretory vesicles, small clear synaptic vesicles (SSVs) and large dense-core vesicles (DCVs), are involved in the secretory process. While they share several common features, a number of differences exist between the exocytosis of these two vesicle types. The focus of the PI's laboratory is the secretory cycle of catecholamine-containing DCVs. As such, we are interested in the elucidation of the molecular machinery that distinguishes DCV and SSV exocytosis. Recent studies have identified CAPS (Ca2+-dependent Activator Protein for Secretion) as a 145 kDa protein that appears to be specific for the DCV secretory pathway. Our preliminary data show that CAPS antagonism exerts a profound inhibitory effect on catecholamine secretion in calf adrenal chromaffin cells. Other studies in permeabilized mammalian cells and synaptosomes show that CAPS plays a role in secretion by DCVs but not SSVs. In this application we propose to analyze in detail the role of CAPS in DCV secretion. There are four specific aims: (1) Determine the stage(s) of the exocytotic pathway regulated by CAPS under physiological conditions. We will use different protocols to isolate the final step of secretion and determine the effect of CAPS antagonism on this step, then determine if CAPS is involved at other stages of the secretory cascade. (2) Determine if CAPS is a Ca2+-sensor for DCV exocytosis and investigate the relationship of CAPS to other candidate Ca sensors like synaptotagmin. (3) Investigate the role of the PH domain of CAPS in its function. (4) Assess whether CAPS function is affected by PKC-dependent phosphorylation and determine if this plays a role in the PKC augmentation of DCV secretion. To accomplish these aims we will use a multidisciplinary approach that includes biochemical, molecular and single-cell electrophysiological methods in chromaffin cells. Techniques such as capacitance and amperometric recordings, that are well-established in the PI's laboratory, will be used to dissect secretory kinetics with millisecond resolution and pinpoint the site of action of CAPS in the exocytotic process. This project will enhance our understanding of CAPS function in exocytosis and illuminate one of the critical differences between the DCV and SSV secretory pathways.